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Abstract: Multiple myeloma (MM) cells are addicted to MYC and its direct transactivation targets IRF4 for proliferation and survival. MYC and IRF4 are still considered "undruggable," as most small-molecule inhibitors suffer from low potency, suboptimal pharmacokinetic properties, and undesirable off-target effects. Indirect inhibition of MYC/IRF4 emerges as a therapeutic vulnerability in MM. Here, we uncovered an unappreciated tumor-suppressive role of C-terminal binding protein 2 (CTBP2) in MM via strong inhibition of the MYC-IRF4 axis. In contrast to epithelial cancers, CTBP2 is frequently downregulated in MM, in association with shortened survival, hyperproliferative features, and adverse clinical outcomes. Restoration of CTBP2 exhibited potent antitumor effects against MM in vitro and in vivo, with marked repression of the MYC-IRF4 network genes. Mechanistically, CTBP2 impeded the transcription of MYC and IRF4 by histone H3 lysine 27 deacetylation (H3K27ac) and indirectly via activation of the MYC repressor IFIT3. In addition, activation of the interferon gene signature by CTBP2 suggested its concomitant immunomodulatory role in MM. Epigenetic studies have revealed the contribution of polycomb-mediated silencing and DNA methylation to CTBP2 inactivation in MM. Notably, inhibitors of Enhance of zeste homolog 2, histone deacetylase, and DNA methyltransferase, currently under evaluation in clinical trials, were effective in restoring CTBP2 expression in MM. Our findings indicated that the loss of CTBP2 plays an essential role in myelomagenesis and deciphers an additional mechanistic link to MYC-IRF4 dysregulation in MM. We envision that the identification of novel critical regulators will facilitate the development of selective and effective approaches for treating this MYC/IRF4-addicted malignancy., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Mitochondrial division inhibitor 1 (Mdivi-1) is a well-known synthetic compound aimed at inhibiting dynamin-related protein 1 (Drp1) to suppress mitochondrial fission, making it a valuable tool for studying mitochondrial dynamics. However, its specific effects beyond Drp1 inhibition remain to be confirmed. In this study, we employed integrative proteomics and phosphoproteomics to delve into the molecular responses induced by Mdivi-1 in SK-N-BE(2)C cells. A total of 3070 proteins and 1945 phosphorylation sites were identified, with 880 of them represented as phosphoproteins. Among these, 266 proteins and 97 phosphorylation sites were found to be sensitive to the Mdivi-1 treatment. Functional enrichment analysis unveiled their involvement in serine biosynthesis and extrinsic apoptotic signaling pathways. Through targeted metabolomics, we observed that Mdivi-1 enhanced intracellular serine biosynthesis while reducing the production of C24:1-ceramide. Within these regulated phosphoproteins, dynamic dephosphorylation of proteasome subunit alpha type 3 serine 250 (PSMA3-S250) occurred after Mdivi-1 treatment. Further site-directed mutagenesis experiments revealed that the dephosphorylation-deficient mutant PSMA3-S250A exhibited a decreased cell survival. This research confirms that Mdivi-1's inhibition of mitochondrial division leads to various side effects, ultimately influencing cell survival, rather than solely targeting Drp1 inhibition.

Strengthening the gut epithelial barrier is a potential strategy for management of gut microbiota-associated illnesses. Here, we demonstrate that dual-specificity phosphatase 6 (Dusp6) knockout enhances baseline colon barrier integrity and ameliorates dextran sulfate sodium (DSS)-induced colonic injury. DUSP6 mutation in Caco-2 cells enhances the epithelial feature and increases mitochondrial oxygen consumption, accompanied by altered glucose metabolism and decreased glycolysis. We find that Dusp6-knockout mice are more resistant to DSS-induced dysbiosis, and the cohousing and fecal microbiota transplantation experiments show that the gut/fecal microbiota derived from Dusp6-knockout mice also confers protection against colitis. Further culturomics and mono-colonialization experiments show that one gut microbiota member in the genus Duncaniella confers host protection from DSS-induced injury. We identify Dusp6 deficiency as beneficial for shaping the gut microbiota eubiosis necessary to protect against gut barrier-related diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vasovagal reaction (VVR) compromises donor safety and reduces the subsequent return rates. Performing applied muscle tension (AMT) during phlebotomy may reduce the incidence of VVR. However, the effectiveness of performing AMT after phlebotomy to reduce delayed VVR remains unclear. With ethics approval, 12 young, first-time donors (YFTD) were recruited to study the effects on stroke volume (SV), cardiac output (CO) and systemic vascular resistance (SVR) while performing AMT from needle insertion to end of recovery. Measurements from 12 matched control YFTD were used for comparison. Pre-donation anxiety and VVR severity were assessed. Compared to controls, donors who performed AMT had higher SV (Control: 57 mL vs. AMT: 69 mL, p = 0.045), higher CO (Control: 3.7 L·min -1 vs. AMT: 5.2 L·min -1 , p = 0.006) and lower SVR (Control: 1962 dyn·s·cm -5 vs. AMT: 1569 dyn·s·cm -5 , p = 0.032) during mid-phlebotomy. During recovery, the AMT group retained higher SV, higher CO and lower SVR than the control, but not reaching statistical significance. Practicing AMT during recovery resulted in sustained haemodynamic improvements beyond the donation period, despite the reduction in delayed VVR was insignificant compared to the control group. A larger sample size is needed to validate the effectiveness of performing AMT after donation to mitigate delayed VVR.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Published by Wiley‐VCH GmbH.)

Background: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown., Methods: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways., Results: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine 1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27 S82 and eIF2a S51 , and triggers autophagosome formation in lung cancer cells., Conclusions: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Cardiac output (CO) monitoring is useful for sports performance training, but most methods are unsuitable as they are invasive or hinder performance. The performance of PhysioFlow (PF), a portable noninvasive transthoracic bioimpedance CO monitor, was evaluated and compared with a reference Doppler CO monitor, USCOM, using a head-up tilt (HUT) test. With ethics committee approval, 20 healthy well-trained athletes were subjected to HUT in a fixed order of 0°, 70°, 30°, and 0° for 3 min each. Simultaneous hemodynamic measurements using PF and USCOM were made 30 s after a change in HUT and analyzed using t tests, ANOVA, and mountain plots. Heart rate (HR) and stroke volume (SV) from both monitors changed according to physiological expectation of tilt, but PF measurements of SV were higher with a positive bias (PF vs. USCOM, 0°: 87.3 vs. 54.0 mL, P < 0.001; 70°: 76.5 vs. 39.5 mL, P < 0.001; 30°: 81.4 vs. 50.1 mL, P < 0.001; 0°: 88.3 vs. 57.1 mL, P < 0.001). Relative changes in SV (∆SV) after each tilt measured using PF were lower with a negative bias (PF vs. USCOM, 0° to 70°: -12.3% vs. -26.3%, P = 0.002; 70° to 30°: +6.4% vs. +31.2%, P < 0.001; 30° to 0°: +9.2% vs. +15.8%, P = 0.280). CO measurements using PF at 70° were erroneous. Compared with USCOM, PF overestimated SV measurements but underestimated the ∆SV between HUT. Accuracy of the PF deteriorated at 70°, implying a gravitational influence on its performance. These findings suggested that the suitability of PF for sports use is questionable. NEW & NOTEWORTHY The use of impedance cardiography to monitor physiological changes in sports is rarely reported. Using head-up tilt test, we evaluated a portable noninvasive impedance cardiography device (PhysioFlow) by comparing it with a reference Doppler monitor (USCOM). Accuracy in tracking hemodynamic changes deteriorated with higher tilt, implying a gravitational influence on its performance. Stroke volume measurements were overestimated, but the changes were underestimated. Despite its convenient physical features, the suitability of PhysioFlow for sports use is questionable.

Gastric cancer is one of the most common types of cancer worldwide. Nevertheless, effective therapeutic strategies have not yet been discovered. Several studies have shown that tanshinone IIA (TIIA), which is extracted from the traditional herbal medicine plant Danshen ( Salvia miltiorrhiza ), has potential activity against many kinds of cancer. Our previous research demonstrated that TIIA can induce cell death in gastric cancer. However, the exact signaling pathway response is still unclear. Post-translational modification (PTM) plays a significant role in a wide range of physiological processes in cancer, via regulation of both signal transduction cascades and many cellular pathways. Here, we integrated multilayer omics-transcriptomics and dynamic phosphoproteomics-to elucidate the regulatory networks triggered by TIIA in gastric cancer. We identified the phosphorylation of heat shock protein 27 (HSP27) at serine 82 in response to TIIA, which caused reactive oxygen species (ROS) production and unfolded protein response (UPR). Moreover, the accumulation of cellular stress increased the expression of heat shock factor 1 (HSF1). In addition, the downstream targets of HSF1, which were involved in heat shock stress and apoptosis, were also activated in TIIA-treated cells. In conclusion, this study performs a multiomic approach to clarify a comprehensive TIIA-responsive network leading to cell death in gastric cancer.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid β-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

MYCN-amplified (MNA) neuroblastoma is an aggressive neural crest-derived pediatric cancer. However, MYCN is indispensable for development and transcriptionally regulates extensive network of genes. Integrating anti-MYCN ChIP-seq and gene expression profiles of neuroblastoma patients revealed the metabolic enzymes, MTHFD2 and PAICS, required for one-carbon metabolism and purine biosynthesis were concomitantly upregulated, which were more susceptible to metastatic neuroblastoma. Moreover, we found that MYCN mediated the folate cycle via MTHFD2, which contributed one-carbon unit to enhance purine synthesis, and further regulated nucleotide production by PAICS in response to cancer progression. Dual knockdown of the MYCN-targeted gene pair, MTHFD2 and PAICS, in MNA neuroblastoma cells synergically reduced cell proliferation, colony formation, migration ability, and DNA synthesis. By systematically screening the compound perturbagens, the gene expression levels of MTHFD2 and PAICS were specifically suppressed by anisomycin and apicidin across cell lines, and our co-treatment results also displayed synergistic inhibition of MNA neuroblastoma cell proliferation. Collectively, targeting a combination of MYCN-targeted genes that interrupts the interconnection of metabolic pathways may overcome drug toxicity and improve the efficacy of current therapeutic agents in MNA neuroblastoma.

Background: Previous studies in Western countries demonstrated BRAF V600E mutation only in a small subset of multiple myeloma (MM) patients. However, the prevalence and clinicopathologic significances of this mutation remain unclear in Chinese MM patients., Patients and Methods: We studied diagnostic bone marrow samples from 205 Chinese MM patients by allele-specific PCR to detect BRAF V600E mutation and by high-resolution melting assay to detect KRAS and NRAS mutations. The mutations were confirmed by independent assays., Results: BRAF V600E mutation was found in 9.3% of the cases, the highest prevalence hitherto reported. In addition, the mutation was significantly associated with hypercalcemia and a male predominance but not with aggressive extramedullary diseases or a high serum creatinine level as reported in Western studies. Importantly, BRAF V600E mutation was an adverse prognostic factor for overall survival in younger MM patients by subgroup analysis. Concurrent analysis of RAS mutations highlighted differential alteration spectrum of RAS signaling between Chinese and Western MM, which may suggest a unique myeloma-related genetic profile in Chinese patients., Conclusion: Our study revealed a higher prevalence of BRAF V600E mutation in Chinese MM patients. The associated prognostic impacts on younger patients could be beneficial to risk stratification and potential application of BRAF-targeted therapies in Chinese MM management. This is the first large-scale study revealing the prevalence and clinicopathologic significances of BRAF V600E mutation in Chinese myeloma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

DNA replication control is vital for maintaining genome stability and the cell cycle, perhaps most notably during cell division. Malignancies often exhibit defective minichromosome maintenance protein 2 (MCM2), a cancer proliferation biomarker that serves as a licensing factor in the initiation of DNA replication. MCM2 is also known to be one of the ATPase active sites that facilitates conformational changes and drives DNA unwinding at the origin of DNA replication. However, the biological networks of MCM2 in lung cancer cells via protein phosphorylation remain unmapped. The RNA-seq datasets from The Cancer Genome Atlas (TCGA) revealed that MCM2 overexpression is correlated with poor survival rate in lung cancer patients. To uncover MCM2-regulated functional networks in lung cancer, we performed multi-dimensional proteomic approach by integrating analysis of the phosphoproteome and proteome, and identified a total of 2361 phosphorylation sites on 753 phosphoproteins, and 4672 proteins. We found that the deregulation of MCM2 is involved in lung cancer cell proliferation, the cell cycle, and migration. Furthermore, HMGA1 S99 phosphorylation was found to be differentially expressed under MCM2 perturbation in opposite directions, and plays an important role in regulating lung cancer cell proliferation. This study therefore enhances our capacity to therapeutically target cancer-specific phosphoproteins.

Lung cancer is the most common cause of cancer-related death worldwide, less than 7% of patients survive 10 years following diagnosis across all stages of lung cancer. Late stage of diagnosis and lack of effective and personalized medicine reflect the need for a better understanding of the mechanisms that underlie lung cancer progression. Quantitative proteomics provides the relative different protein abundance in normal and cancer patients which offers the information for molecular interactions, signaling pathways, and biomarker identification. Here we introduce both theoretical and practical applications in the use of quantitative proteomics approaches, with principles of current technologies and methodologies including gel-based, label free, stable isotope labeling as well as targeted proteomics. Predictive markers of drug resistance, candidate biomarkers for diagnosis, and prognostic markers in lung cancer have also been discovered and analyzed by quantitative proteomic analysis. Moreover, construction of protein networks enables to provide an opportunity to interpret disease pathway and improve our understanding in cancer therapeutic strategies, allowing the discovery of molecular markers and new therapeutic targets for lung cancer.